Spatial genomic tools to interrogate T cell clonotypes, tumor clones and the microenvironment

免疫疗法的当代临床成功突出了肿瘤浸润细胞的关键作用 在介导抗肿瘤免疫中的作用，并且通常将肿瘤内T细胞的存在与 治疗反应。然而，到目前为止，用于评估T细胞状态，其克隆 身份和本地化是不可能的。近年来，吴实验室已经取得了几项成果， 显著的技术进步，包括产生最佳的HLA I类表位预测因子（HLA抗原）; 用于单细胞TCR测序的高度稳健的基于靶向板的方法（rhTCRseq）;以及 并行克隆数百个TCR，使得它们可以被询问以确定性地将TCR与其受体连接。 抗原特异性因为肿瘤反应性T细胞和恶性克隆之间的动态相互作用发生 在定义的组织空间排序中，我们有价值的新见解促使我们研究空间 肿瘤特异性T细胞的组织涉及肿瘤克隆的原位定位。我们假设抗原 特异性驱动T细胞和肿瘤细胞之间的相互作用，影响不同的区域定位， T细胞在肿瘤微环境中的基线和治疗背景下;相反， 的空间定位鉴定T细胞克隆特异性不同的抗原类型。Slide-seq技术，由 Chen实验室提供了一个易于处理和令人兴奋的途径，通过实施一个可扩展的 方法进行深入分析的信息人类和小鼠肿瘤组织。通过扩大 这种无偏的细胞分辨率空间捕获方法的能力，我们的目标是获得组织水平的理解 T细胞克隆的丰度和功能状态以及它们在肿瘤中的空间定位 微环境是相通的。特别是，该研究将解决T细胞克隆的空间组织， 人和小鼠肿瘤中的肿瘤亚克隆。我们的技术目标将是提高 转录本捕获的技术，扩展能力，包括强大的检测肿瘤突变， 开发一套分析工具，以多模型方式整合转录本、DNA水平和TCR水平， 信息（目标1-2）。针对RCC肿瘤，我们将评估T细胞的细胞-细胞定位模式， 通过TCR和DNA的整合空间分析确定与肿瘤亚克隆和基质细胞相关的克隆 Slide-seq数据（目标2）。最后，我们将评估抗原特异性的影响（通过稳健的 我们实验室建立的TCR重建和询问方法）对T细胞表型和定位的影响， 基线和免疫检查点阻断后的TCR和DNA Slide-seq整合空间分析， 新抗原疫苗(Aim 3）总之，我们将开发和测试这些工具，以了解空间定位 驱动免疫反应的细胞网络，以及T细胞受体与肿瘤的关系 微环境我们工作的完成将产生一个全面的工具集， 肿瘤免疫反应的细胞和组织学理解。

The contemporary clinical successes of immunotherapies have highlighted the key role of tumor-infiltrating cells in mediating anti-tumor immunity and have generally associated the presence of T cells within tumors with therapeutic response. However, until now, a systematic approach for evaluating how T cell state, their clonal identity and localization are related has not been possible. In recent years, the Wu lab has achieved several notable technical advances, including generation of a best-in-class HLA class I epitope predictor (HLAthena); a highly robust targeted plate-based method for single-cell TCR sequencing (rhTCRseq); and a means to parallelize the cloning of hundreds of TCRs such that they can be interrogated to definitively link a TCR with its antigen specificity. Because dynamic interactions between tumor-reactive T cells and malignant clones occur within the defined spatial ordering of tissue, our valuable new insights motivate us to investigate how the spatial organization of tumor-specific T cells relates to in situ positioning of tumor clones. We hypothesize that antigen specificity, which drives the interactions between T cells and tumor cells, impacts the distinct regional localization of T cells within the tumor microenvironment at baseline and in the context of therapy; conversely, that knowledge of spatial localization identifies T cell clones specific for distinct antigen types. Slide-seq technology, created by the Chen lab, provides a tractable and exciting path to investigate this hypothesis by implementing a scalable approach to undertake in-depth analyses of informative human and murine tumor tissues. By expanding the capabilities of this unbiased cellular resolution spatial capture method, we aim to gain tissue level understanding of how the abundance and functional state of T cell clones and their spatial orientation within the tumor microenvironment are linked. In particular, the study will address the spatial organization of T cell clones and tumor subclones in human and mouse tumors. Our technology goals will be to increase the efficiency of transcript capture of the technology, extend the capability to include robust detection of tumor mutations, and to develop a suite of analytic tools to integrate in a multi model fashion the transcript, DNA-level and TCR levels of information (Aims 1-2). Focusing on RCC tumors, we will evaluate the cell-cell localization patterns of T cell clones in relation to tumor subclones and stromal cells through integrated spatial analyses of TCR and DNA Slide-seq data (Aim 2). Finally, we will evaluate the impact of antigen specificity (definitively assessed by robust TCR reconstruction and interrogation methods established in our lab) on T cell phenotype and localization using TCR and DNA Slide-seq integrated spatial analyses at baseline and following immune checkpoint blockade and neoantigen vaccine. (Aim 3) Altogether, we will develop and test these tools to understand spatially localized cellular networks which drive immune response, and T-cell receptor relationships with the tumor microenvironment. The completion of our work will yield a comprehensive toolset to enable a molecular, cellular and histological understanding of the tumor immune response.